MIST,a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.     

Structure analysis of Entamoeba histolytica DNMT2 (EhMeth)

In eukaryotes, DNA methylation is an important epigenetic modification that is generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2 family have been shown to have a dual substrate specificity acting on DNA as well as on three specific tRNAs (tRNA(Asp), tRNA(Val), tRNA(Gly)). Entamoeba histolytica is a major human pathogen, and expresses a single DNA MTase (EhMeth) that belongs to the DNMT2 family and shows high homology to the human enzyme as well as to the bacterial DNA MTase M.HhaI. The molecular basis for the recognition of the substrate tRNAs and discrimination of non-cognate tRNAs is unknown. Here we present the crystal structure of the cytosine-5-methyltransferase EhMeth at a resolution of 2.15 ?, in complex with its reaction product S-adenosyl-L-homocysteine, revealing all parts of a DNMT2 MTase, including the active site loop. Mobility shift assays show that in vitro the full length tRNA is required for stable complex formation with EhMeth.     

The mechanism of action of tRNA methylases studied with immobilized tRNAs.

Each of the individual tRNAs immobilized on aminohydroxybutyl-cellulose (ABC) through their oxidized 3'-terminal binds affinitively all methylases present in the enzyme extract irrespective of whether this tRNA will be involved in the following step of methylation or not. These data allow to suggest that (a) the formation of a methylase-tRNA complex and the catalytic act of methylation are indeed autonomous processes and (b) the first step of interaction between tRNAs and tRNA methylases is rather unspecific and consists in the recognition of the whole class of tRNA molecules.     

Evidence for two types of complexes formed by yeast tyrosyl-tRNA synthetase with cognate and non-cognate tRNA. Effect of ribonucleoside triphosphates

Polyacrylamide gel electrophoresis at pH 8.3 was used to detect and quantitate the formation of the yeast tyrosyl-tRNA synthetase (an alpha 2-type enzyme) complex with its cognate tRNA. Electrophoretic mobility of the complex is intermediate between the free enzyme and free tRNA; picomolar quantities can be readily detected by silver staining and quantitated by densitometry of autoradiograms when [32P]tRNA is used. Two kinds of complexes of Tyr-tRNA synthetase with yeast tRNA(Tyr) were detected. A slower-moving complex is formed at ratios of tRNA(Tyr)/enzyme less than or equal to 0.5; it is assigned the composition tRNA.(alpha 2)2. At higher ratios, a faster-moving complex is formed, approaching saturation at tRNA(Tyr)/enzyme = 1; any excess of tRNA(Tyr) remains unbound. This complex is assigned the composition tRNA.alpha 2. The slower, i.e. tRNA.(alpha 2)2 complex, but not the faster complex, can be formed even with non-cognate tRNAs. Competition experiments show that the affinity of the enzyme towards tRNA(Tyr) is at least 10-fold higher than that for the non-cognate tRNAs. ATP and GTP affect the electrophoretic mobility of the enzyme and prevent the formation of tRNA.(alpha 2)2 complexes both with cognate and non-cognate tRNAs, while neither tyrosine, as the third substrate of Tyr tRNA synthetase, nor AMP, AMP/PPi, or spermidine, have such effects. Hence, the ATP-mediated formation of the alpha 2 structure parallels the increase in specificity of the enzyme towards its cognate tRNA.     

A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica

Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix–turn–helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix α1 contacts tRNA whereas helix α2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.     

Aminoacyl-tRNA synthetase-induced cleavage of tRNA.

Aminoacyl-tRNA synthetases interact with their cognate tRNAs in a highly specific fashion. We have examined the phenomenon that upon complex formation E. coli glutaminyl-tRNA synthetase destabilizes tRNA(Gln) causing chain scissions in the presence of Mg2+ ions. The phosphodiester bond cleavage produces 3'-phosphate and 5'-hydroxyl ends. This kind of experiment is useful for detecting conformational changes in tRNA. Our results show that the cleavage is synthetase-specific, that mutant and wild-type tRNA(Gln) species can assume a different conformation, and that modified nucleosides in tRNA enhance the structural stability of the molecule.     

G-Domain Dimerization Orchestrates the tRNA Wobble Modification Reaction in the MnmE/GidA Complex

MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation between MnmE and GidA involves large conformational changes that induce G-domain dimerization of MmnE and that GidA co-stimulates GTP hydrolysis on MnmE. Starting from a structural model of the complex, we identify interface mutations disrupting complex formation or communication. Although GidA does not directly contact the G-domains, conformational changes in MnmE, induced by G-domain dimerization in the triphosphate state, regulate the affinity for GidA. We developed a tRNA modification assay and demonstrate for the first time in vitro that the MnmE/GidA complex catalyzes incorporation of glycine into tRNA. An intact MnmE/GidA complex rather than their sequential action is crucial for in vitro modification. Since only GTP, but not GDP or non-hydrolyzable GTP analogs, drives the MnmE/GidA-catalyzed modification reaction, we conclude that GTP hydrolysis is essential for activity. We finally show that an active GTPase, an intact MnmE/GidA communication, and dimerization of G-domains are necessary for in vivo functioning since mutations disrupting either result in a respiratory deficient phenotype in yeast.     

Kinetically competent intermediates in the translocation step of protein synthesis

Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.     

The unusual methanogenic seryl-tRNA synthetase recognizes tRNASer species from all three kingdoms of life.

The methanogenic archaea Methanococcus jannaschii and M. maripaludis contain an atypical seryl-tRNA synthetase (SerRS), which recognizes eukaryotic and bacterial tRNAsSer, in addition to the homologous tRNASer and tRNASec species. The relative flexibility in tRNA recognition displayed by methanogenic SerRSs, shown by aminoacylation and gel mobility shift assays, indicates the conservation of some serine determinants in all three domains. The complex of M. maripaludis SerRS with the homologues tRNASer was isolated by gel filtration chromatography. Complex formation strongly depends on the conformation of tRNA. Therefore, the renaturation conditions for in vitro transcribed tRNASer(GCU) isoacceptor were studied carefully. This tRNA, unlike many other tRNAs, is prone to dimerization, possibly due to several stretches of complementary oligonucleotides within its sequence. Dimerization is facilitated by increased tRNA concentration and can be diminished by fast renaturation in the presence of 5 mm magnesium chloride.     

An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.     

EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome.

Translocation, catalyzed by elongation factor EF-G, is the precise movement of the tRNA-mRNA complex within the ribosome following peptide bond formation. Here we examine the structural requirement for A- and P-site tRNAs in EF-G-catalyzed translocation by substituting anticodon stem-loop (ASL) analogs for the respective tRNAs. Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting. Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA-dependent fashion. The requirement for an A-site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop. Translocation of the ASL is both EF-G- and GTP-dependent, and is inhibited by the translocational inhibitor thiostrepton. These findings show that the D, T and acceptor stem regions of A-site tRNA are not essential for EF-G-dependent translocation. In contrast, no translocation occurs if the P-site tRNA is substituted with an ASL, indicating that other elements of P-site tRNA structure are required for translocation. We also tested the effect of increasing the A-site ASL stem length from 4 to 33 bp on translocation from A to P site. Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon-D arm axis. This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon-D arm.     

Yeast aspartyl-tRNA synthetase residues interacting with tRNA(Asp) identity bases connectively contribute to tRNA(Asp) binding in the ground and transition-state complex and discriminate against non-cognate tRNAs.

Crystallographic studies of the aspartyl-tRNA synthetase-tRNA(Asp)complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNA(Asp). Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNA(Asp)recognition and acylation. The results showed that contacts with the acceptor A of tRNA(Asp)by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNA(Asp). Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state. These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis. This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site. This signal might be conveyed via tRNA(Asp)as suggested by the observed conformational change of this molecule upon interaction with AspRS. From binding free energy values corresponding to the different AspRS-tRNA(Asp)interaction domains, it might be concluded that upon complex formation, the anticodon interacts first. Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNA(Asp)and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs.     

Elongation factors on the ribosome

The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.     

Mechanism of the difference in the binding affinity of E. coli tRNAGln to glutaminyl-tRNA synthetase caused by noninterface nucleotides in variable loop

Aminoacyl-tRNA synthetases (ARSs) distinguish their cognate tRNAs from many other kinds of tRNAs, despite the very similar tertiary structures of tRNAs. Many researchers have supported the view that this recognition is achieved by intermolecular interactions between tRNA and ARS. However, one of the aptamers of Escherichia coli glutamine specific tRNA, var-AGGU, has a higher affinity to ARS than the wild-type, although the sequence difference only lies in the variable loop located on the opposite side of the binding interface with ARS. To understand the reason for the difference in affinity, we did molecular dynamics simulations on tRNAs and their complexes with ARS. We calculated the enthalpic and entropic contributions to the binding free energy with the molecular mechanics-Poisson-Boltzmann/surface area method and found that the entropic difference plays an important role in the difference in binding free energies. During the molecular dynamics simulations, dynamic rearrangements of hydrogen bonds occurred in the tertiary core region of the wild-type tRNA, whereas they were not observed in the free var-AGGU simulation. Since the internal mobility was suppressed upon complex formation with ARS, the entropy loss in the wild-type was larger than that of the aptamer. We therefore concluded that the sequence difference in the variable loop caused the difference in the internal mobility of the tertiary core region tRNAs and led to the difference in the affinity to ARS through the entropy term.     

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 5' end of anticodon stem-loop analogs (ASLs) of tRNA and to the 5' end of deacylated tRNA(Phe) to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 45 degrees between the respective planes of the molecules. The terminal phosphates of 3'CCA are separated by 23 A when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.     

A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity.

Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNA(Gln) binding to glnRS/ATP complex is absent in a noncognate tRNA tRNA(Glu)-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNA(Gln)-induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.     

How U38, 39, and 40 of many tRNAs become the targets for pseudouridylation by TruA

Translational accuracy and efficiency depend upon modification of uridines in the tRNA anticodon stem loop (ASL) by a highly conserved pseudouridine synthase TruA. TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures through a poorly characterized mechanism that differs from previously studied RNA-modifying enzymes. The molecular basis for the site and substrate "promiscuity" was studied by determining the crystal structures of E. coli TruA in complex with two different leucyl tRNAs in conjunction with functional assays and computer simulation. The structures capture three stages of the TruA*tRNA reaction, revealing the mechanism by which TruA selects the target site. We propose that TruA utilizes the intrinsic flexibility of the ASL for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function.     

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding

Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.     

Pathogenic mutations in antisense mitochondrial tRNAs

Pathogenic mutations in mitochondrial tRNAs are 6.5 times more frequent than in other mitochondrial genes. This suggests that tRNA mutations perturb more than one function. A potential additional tRNA gene function is that of templating for antisense tRNAs. Pathogenic mutations weaken cloverleaf secondary structures of sense tRNAs. Analyses here show similar effects for most antisense tRNAs, especially after adjusting for associations between sense and antisense cloverleaf stabilities. These results imply translational activity by antisense tRNAs. For sense tRNAs Ala and Ser UCN, pathogenicity associates as much with sense as with antisense cloverleaf formation. For tRNA Pro, pathogenicity seems associated only with antisense, not sense tRNA cloverleaf formation. Translational activity by antisense tRNAs is expected for the 11 antisense tRNAs processed by regular sense RNA maturation, those recognized by their cognate amino acid’s tRNA synthetase, and those forming relatively stable cloverleaves as compared to their sense counterpart. Most antisense tRNAs probably function routinely in translation and extend the tRNA pool (extension hypothesis); others do not (avoidance hypothesis). The greater the expected translational activity of an antisense tRNA, the more pathogenic mutations weaken its cloverleaf secondary structure. Some evidence for RNA interference, a more classical role for antisense tRNAs, exists only for tRNA Ser UCN. Mutation pathogenicity probably frequently results from a mixture of effects due to sense and antisense tRNA translational activity for many mitochondrial tRNAs. Genomic studies should routinely explore for translational activity by antisense tRNAs.